A proteolytic pathway that has been implicated to play a major role in selective degradation of abnormal and short-lived proteins requires the covalent attachment of ubiquitin to a target protein prior to degradation (for recent reviews see Finley and Chau (1991) Annu. Rev. Cell Biol. 7, 25-69; Jentsch et al.(1991) Biochim. Biophys. Acta 1089, 127-139; and Hershko and Ciechanover (1992) Annu. Rev. Biochem. 61, 761-807). Ubiquitin is a highly conserved protein of 76 amino acids found only in eukaryotes. The basic enzymatic reactions involved in the ubiquitination of proteins are well characterized. Ubiquitin is first activated by the E1 enzyme in an ATP-dependent step in which a thioester is formed between the active site cysteine residue of E1 and the C-terminal glycine of ubiquitin. The activated ubiquitin is transferred to a cystein residue of ubiquitin-conjugating (E2) enzymes, which then catalyze the formation of an isopeptide bond between the C-terminal glycine of ubiquitin and the .epsilon.-amino group of a lysine residue on a target protein. Ubiquitin also becomes conjugated to itself via a lysine residue at position 48 of ubiquitin, resulting in the formation of multiubiquitin chains (Chau et al. (1989) Science 243, 1576-1583). Finally, multiubiquitinated proteins serve as targets that are recognized and degraded by an ATP-dependent protease complex.
Some members of the E2 family are able to ubiquitinate certain proteins in the absence of any additional proteins, whereas others require the presence of ubiquitin-protein ligases (E3s). E3s are defined as proteins that participate with E1 and E2s in the ubiquitination of proteins, presumably by specifically binding to target proteins that are otherwise not recognized by E2s (Hershko and Ciechanover (1992) Annu. Rev. Biochem. 61, 761-807). Although only a few E3s have been described (Rolfe et al. (1995) PNAS 92:3264-3268; King et al. (1995) Cell 81:279-288; Reiss and Hershko (1990) J. Biol. Chem. 265, 3685-3690; Heller and Hershko (1990) J. Biol. Chem. 265, 6532-6535; and Bartel et al. (1990) EMBO J. 9, 3179-3189), the existence of a large group of proteins possessing E3-like activity is implied by the fact that selective protein degradation requires the specific targeting of may different proteins, often at particular stages of the cell cycle or differentiation.
The enzymatic reactions of the ubiquitin-dependent proteolytic pathway have been elucidated using damaged or artificial proteins. Only a few normal cellular targets of the ubiquitin system have been identified. These include plant phytocharome, the yeast transcription factor MAT .alpha.2, and cyclin B (Jabben et al. (1989) J. Biol. Chem. 264, 4998-5005; Hochstrasser et al. (1991) Proc. Natl. Acad. Sci. USA 88, 4606-4610; Glotzer et al.(1991) Nature 349, 132-138). The relevant E2s or E3s that are involved in the ubiquitination of these proteins have yet to be identified.
A novel mechanism of action for dominant oncogenes was postulated based on the observation that E6 protein of the human papillomavirus (HPV) types 16 and 18 could stimulate the ubiquitin-dependent degradation of the p53 tumor suppressor protein in vitro (Scheffner et al. (1990) Cell 63, 1129-1136).
Certain HPV types, such as HPV-16 and HPV-18, that infect the anogenital tract are associated with malignant lesions, most notably cervical cancer (for review see zur Hausen, (1991) Virology 184, 9-13). These types are referred to as high risk HPVs, as opposed to low risk anogenital-specific HPVs such as HPV-6 and HPV-11, which are generally associated only with benign lesions such as condyloma accuminata. Various cellular transformation assays have shown that the high risk HPVs encode two oncoproteins, E6 and E7. Significant to cervical carcinogenesis, both E6 and E7 are necessary and sufficient for efficient immortalization of human squamous epithelial cells, which are the natural host cells of these viruses (Munger et al. (1989) J. Virol. 63, 4417-4421; Hawley-Nelson et al. (1989) EMBO J. 8, 3905-3910) and both of these genes are expressed in HPV-positive cervical cancers. Insight into the mechanisms by which E6 and E7 may contribute to cellular transformations has come from the recognition that, similar to the oncoproteins of other small DNA tumor viruses, E6 and E7 bind to cell regulatory proteins. Like the simian virus 40 large tumor antigen (SV40 T antigen) and the adenovirus 5 (Ad5) E1A proteins, E7 can bind to the product of the tumor suppressor gene RB (DeCaprio et al. (1988) Cell 54, 275-283; Whyte et al. (1988) Nature 334, 124-129; and Dyson et al. (1989) Annu. Rev. Cell Biol. 7, 25-69). The E6 protein, the SV40 T antigen, and the Ad5 E1B 55 kd protein are able to complex with wild-type p53 (Wemess et al. (1990) Science 248, 76-79; Lane and Crawford (1979) Nature 351, 453-456; Linzer and Levine (1979) Cell 17, 43-52; and Sarnow et al. (1982) Cell 28, 387-394). Mutations within the p53 gene, presumably inactivate the tumor suppressor function of wild-type p53 (Scheffner et al. (1991) Proc. Natl. Acad. Sci. USA 88, 5523-5527; Crook et al. (1991) Oncogene 6, 873-875; and Lin and Simmons (1990) Virology 176, 302-305). This is consistent with the hypothesis that complex formation with the viral oncoproteins interferes with the negative growth regulatory function of p53 and pRB. The mechanisms, however, by which the viral oncoproteins inactivate p53 seem to be quite different. The level and the half-life of p53 in SV40 and adenovirus-transformed cells are increased compared with uninfected cells (Oren et al. (1981) Mol. Cell. Biol. 1, 101-110; Recich et al. (1983) Mol. Cell. Biol. 3, 2143-2150), suggesting that SV40 T antigen and the Ad5 E1B 55 kd protein inactivate p53 by sequestering it into stable complexes. In contrast, the level and half-life of p53 in E6 immortalized cell lines or in HPV-positive cervical carcinoma cells are generally decreased (Scheffner et al. (1991) Proc. Natl. Acad. Sci USA 88, 5523-5527; Hubbert et al. (1992) J. Virol. 66, 6237-6241). This observation is consistent with the in vitro experiments showing that E6 stimulates the degradation of p53 via the ubiquitin-dependent proteolytic system (Scheffner et al. (1990) Cell 63, 1129-1136).
Attempts to study the binding of E6 to p53 in the absence of subsequent degradation revealed an additional cellular protein of an approximate molecular size of 100 kDa that participates in a complex with E6 (Huibregtse et al. (1991) EMBO J. 13, 4129-4135). This 100 kDa protein can interact with high risk HPV E6 proteins in the absence of p53, but not with p53 in the absence of E6 proteins, and was therefore termed E6-associated protein (E6AP). Recently, it has been report that the E6/E6AP complex, in concert with the ubiquitin-activating enzyme E1 and an E2, can mediate the ubiquitination of p53 in vitro (Scheffner et al. (1993) Cell 75, 495-505). This indicates that in this system the complex formed with E6, which can include E6AP, has the activity of a ubiquitin-protein ligase.